Understanding of human intestinal physiology, pathophysiology of diarrheal diseases and development of anti-diarrheal drug therapy has been held back by lack of an available and easy to use model of human intestine. Recently two methods (from LGR5 positive intestinal stem cells and from isolated intact intestinal crypts) have been developed to allow development of human small intestinal organoids that form normal appearing villus/crypt axes which contain all normally occurring epithelial cells (Na absorptive, Cl secretory, goblet, enteroendocrine and Paneth). We propose to compare the two types of human small intestinal organoids and use them to advance understanding of normal intestinal digestive physiology and the pathophysiology of several important human diarrheal diseases. We will 1) compare the two types of human small intestinal organoids for a) how they develop polarity and differentiation over time and define the distribution and changes with organoid differentiation of transport proteins involved in Na absorption and Cl secretion, processes which become abnormal in diarrhea. b) Test the function in the organoids of two transport proteins that are important for the pathophysiology of diarrhea, NHE3 and CFTR under basal and regulated conditions which mimic normal digestive physiology and become abnormal in diarrhea. c) Use the organoids and understanding of Na absorption and Cl secretion to increase understanding of three important human diarrheal diseases caused by cholera toxin, rotavirus and enterohemorrhagic E. coli. Other important potential uses of the organoids are for a) high throughput drug screening based on the fluorescence assays for NHE3 and CFTR and b) personalized medicine, since the organoids can be developed from individual patients. PUBLIC HEALTH RELEVANCE: Understanding of human intestinal physiology, pathophysiology of diarrheal diseases and development of anti-diarrheal drug therapy has been held back by lack of an available and easy to use model of human intestine. There are now two methods allowing growth of intestinal organoids which consist of all normally present intestinal epithelial cells organized into a crypt/villus axis. We will compare these two methods as to how they polarize, differentiate, and the location of transport proteins the abnormal function of which contribute to the pathophysiology of diarrhea (NHE3 and CFTR); evaluate the function of these transport proteins under normal conditions; and use the organoid models to study pathophysiology, including function of these transport proteins, of three important diarrheal diseases, cholera, rotavirus and enterohemorrhagic E. coli.